For prevention and cure of AIDS, it is important to know the mechanism of how cell lineage for T lymphocyte is determined during embryonic development. T lymphocytes are derived from hematopoietic stem cells, however, it is still unclear how hematopoietic cells are developed from undifferentiated mesodermal tissues. We are focusing on the function of Bone Morphogenetic Proteins (BMPs), the members of TGF-beta superfamily, during mouse development. BMPs have function to specify the fate of mesodermal tissues more ventral. Hematopoietic cells are derived from the most ventral mesoderm, therefore, BMPs are hypothesized to play important roles during specification of T lymphocyte lineage in the hematopoietic tissues. One of the difficulties in studying mouse development is that embryogenesis occurs inside of the mother. To overcome this problem, we are trying to establish in vitro culture system for whole embryos. The tissue-specific gene targeting method described in Project ES71004-02 is a powerful technique to address the function of the given genes in given tissues. However, it still has limitations dependent upon the specificity and strength of promoters to express Cre recombinase. Another goal of this project is to overcome this limitation. The basic strategy is to culture embryos that have floxed alleles using whole embryo culture system, and then to microinject recombinant Adenovirus to express Cre recombinase in the interested region of embryos. Through this method, we could achieve region specific gene targeting regardless of the specificity of promoters. First, we established the culture method for the embryos as early as day 5.5 embryos. We maintain a transgenic mouse lines that expresses lacZ depending on the DNA recombination catalyzed Cre recombinase. As a model system, we culture embryos taken from this transgenic line and inject Cre expressing Adenovirus (Adex-Cre). We injected Adex-Cre into the amniotic cavity of day 7.5 embryos to get lacZ staining. However, population of the cells that expressed lacZ was very low. We are underway to optimize the experimental condition. Once this condition is established, we will apply this method for neuroectoderm specific disruption of receptors for BMPs. In addition to the region specific injection of the Cre expressing virus, we will add the virus to the culture media to infect the yolk sac, the most outer layer of cultured embryos. The yolk sac is the organ for primitive hematopoiesis, starting at day 7.5 of gestation. There is debate whether the origin of definitive hematopoiesis starts at day 9.5 is derived from the yolk sac. We could address this question by the disruption of Bmpr in yolk sac-specific manner. The knowledge that will be acquired through this research may give us better understanding for the primitive and definitive hematopoiesis and that may lead some insights for prevention and cure of AIDS.